The overall objective of this proposal is to provide better insight into mechanisms that cause a-cell apoptosis in Type 1, insulin-dependent diabetes mellitus (IDDM). Although the autoimmune etiology of IDDM is well established, a-cell death signaling mechanisms are incompletely understood. Endoplasmic reticulum (ER) stress induced by disruption of ER Ca2+ homeostasis causes apoptosis in MIN6 cells and in mouse islets of Langerhans. The molecular signals linking ER stress and a-cell death are unresolved. The role of ER stress in IDDM has not been defined. Apoptosis induced by ER stress may be related to activation of caspases, a family of cysteine proteases, and increased expression of the transcription factor CHOP (C/EBP homologous protein also known as GADD153) gene. Preliminary experiments suggest that sarcoendoplasmic reticulum calcium ATPase (SERCA), a key regulator of ER Ca2+ homeostasis, is impaired in islets from non-obese diabetic (NOD) mice, a model of IDDM. This raises an intriguing possibility that loss of functional a-cell mass in diabetes is related to ER stress. The proposed experiments will: [1] identify molecular signals induced by ER stress in a-cells; [2] determine whether cytokines induce beta-cell ER stress; and [3] define the relationship between ER stress and loss of functional a-cell mass in NOD mice. The following hypotheses will be tested: [1] ER stress activates beta-cell apoptosis via caspase- and CHOP-dependent pathways; [2] ER stress induces mitochondrially-derived cell death signals; [3] cytotoxic cytokines perturb beta-cell ER Ca2+ homeostasis, activate caspases and induce CHOP expression; and [4] development of diabetes in NOD mice is correlated with ER stress. MIN6 cells and mouse islet cells will be studied. Experimental procedures will include visualization of subcellular Ca2+ concentration gradients with genetically targeted Ca2+ biosensors, measurements of caspase activity, and application of Laser Capture Microdissection and real-time PCR to monitor SERCA and CHOP expression in islets. The proposed studies will provide new insights into mechanisms regulating beta-cell viability and facilitate development of novel therapeutic strategies to preserve and maintain functional beta-cell mass in patients with Type I diabetes.